Pharmacokinetic enhancements of bifunctional chelates and uses thereof

ABSTRACT

The present invention relates to conjugates including a chelating moiety of a metal complex thereof and a therapeutic or targeting moiety, methods for their production, and uses thereof.

RELATED APPLICATIONS

The present application claims priority to, and the benefit of, U.S.Provisional Patent Application No. 62/502,260 titled “PHARMACOKINETICENHANCEMENTS OF BIFUNCTIONAL CHELATES AND USES THEREOF” and filed on May5, 2017, the entire contents of which are hereby incorporated byreference for all purposes.

BACKGROUND

Radiolabelled targeting moieties, or radioconjugates, are typicallyprepared by using a bifunctional chelator to append a radiolabel to abiological molecule while maintaining target affinity. Bifunctionalchelates structurally may contain a chelate, a linker, and across-linking group or targeting moiety. By modifying the linker regionof the bifunctional chelate, pharmacokinetic advantages can be obtainedthat can increase the excretion of radioactivity.

SUMMARY OF THE INVENTION

The present invention is directed to linkers that enhance the excretionof a chelating moiety, or a metal complex thereof, when conjugated to atherapeutic moiety, a targeting moiety, or a cross-linking group.

Accordingly, in a first aspect, the invention features a compound havingthe structure:

A-L¹-(L₂)_(n)-B   Formula I

wherein A is chelating moiety or a metal complex thereof;

L¹ is optionally substituted C₁-C₆ alkyl, substituted C₁-C₆ heteroalkyl,substituted aryl or heteroaryl;

B is a therapeutic moiety, a targeting moiety, or cross-linking group,

or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L², independently, has the structure:

(—X¹-L³-Z¹—)   Formula II

wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹),—CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹) , O, NR¹ and R¹ is H or optionallysubstituted C₁-C₆ alkyl or optionally substituted C₁-C₆ heteroalkyl,substituted aryl or heteroaryl; L³ is optionally substituted C₁-C₅₀alkyl or optionally substituted C₁-C₅₀ heteroalkyl or C₅-C₂₀polyethylene glycol; Z¹ is CH₂, C═O, C═S, OC═O, NR¹C═O, NR¹ and R¹ is ahydrogen or optionally substituted C₁-C₆ alkyl, pyrrolidine-2,5-dione.

In some embodiments, the chelating moiety is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α, α′, α″,α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane),DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid),DO3AM-acetic acid(2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)aceticacid), DOTA-GA anhydride(2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-tril)triaceticacid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP(1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid,DOTA-4AMP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonicacid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP(1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA(1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid),PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaaceticacid), H₄Octapa(N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diaceticacid), H₂Dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane),H₆phospa(N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane),TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P(tetraazacyclododecane dimethanephosphonic acid), HP-DO3A(hydroxypropyltetraazacyclododecanetriacetic acid), EDTA(ethylenediaminetetraacetic acid), Deferoxamine, DTPA(diethylenetriaminepentaacetic acid), DTPA-BMA(diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentatehydroxypyridinones) or porphyrin.

The person having ordinary skill in the art will understand that the useof chelating moieties in the practice of the invention are not limitedto the specific constructs disclosed herein, but rather may includeother known chelating moieties.

In some embodiments, the chelating moiety has the structure:

wherein Y¹ is —CH₂OCH₂(L²)_(n)-B, C═S(L²)_(n)-B, or C═S(L²)_(n)-B and Y²is —CH₂CO₂H;

wherein Y¹ is H, Y² is L¹-(L²)_(n)-B

In some embodiments, L¹ has the structure:

wherein R² is optionally substituted hydrogen or —CO₂H

In some embodiments, the metal can be selected from Bi, Pb, Y, Mn, Cr,Fe, Co, Zn Ni, Tc, In, Ga, Cu, Re, Sm, a lanthanide, or an actinide, foruse as imaging or therapeutic agents. Specific examples of radionuclidessuitable for complexing to a compound of formula (I) include ⁴⁷Sc, ⁵⁵Co,⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Pb, ²¹²Pp, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th.

In some embodiments, B is a therapeutic moiety or targeting moiety.

In some embodiments, the therapeutic moiety or targeting moiety is anantibody, an antigen-binding fragment thereof or other targeting proteinsuch as nanobodies, affibodies, and consensus sequences from Fibronectintype III domains.

In some embodiments, the antibody, or an antigen-binding fragmentthereof specifically binds insulin-like growth factor-1 receptor(IGF-1R).

In some embodiments, the cross-linking group is an amino-reactive, amethionine-reactive cross linking group, a thiol-reactive cross-linkinggroup or a sortase-mediated coupling sequence.

In some embodiments, the amino-reactive, a methionine-reactive orthiol-reactive cross-linking group comprises an activated ester such asa hydroxysuccinimide ester, N-hydroxysulfosuccinimide,2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate,anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne,strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide,diazirine, phosphine, tetrazine, isothiocyanate, or an oxaziridine.

In some embodiments, the sortase recognition sequence may comprise of aterminal glycine-glycine-glycine (GGG) and/or LPTXG amino acid sequence,where X is any amino acid.

The person having ordinary skill in the art will understand that the useof cross linking groups in the practice of the invention are not limitedto the specific constructs disclosed herein, but rather may includeother known cross linking groups.

In some embodiments, the cross-linking group is selected from the groupconsisting of:

In some embodiments, Y′ is H.

In some embodiments, X¹ is C═O(NR¹) and R¹ is H.

In some embodiments, Z′ is —CH₂.

In some embodiments, L² has n value of 1.

In some embodiments, the compound is selected from the group consistingof:

In some embodiments, the metal is a radionuclide.

In some embodiments, the radionuclide is ¹¹¹In.

In some embodiments, the radionuclide is ⁶⁸Ga.

In some embodiments, the radionuclide is ⁸⁶Y.

In some embodiments, the metal is a beta-emitting radionuclide.

In some embodiments, the radionuclide are ⁶⁷Cu, ¹⁷⁷Lu′ or ⁹⁰Y

In some embodiments, the metal is an alpha-emitting radionuclide. Insome embodiments, the radionuclide is ²²⁵Ac, ²¹²Pb, ²²⁷Th or the progeny(daughter isotopes) thereof.

In another aspect, the invention features a pharmaceutical compositionincluding any of the foregoing compounds and a pharmaceuticallyacceptable excipient.

In another aspect, the invention features a method of radiationtreatment planning and/or radiation treatment, the method comprisingadministering to a subject in need thereof any of the foregoingcompounds or pharmaceutical compositions.

In another aspect, the invention features a method of detecting and/ortreating cancer, the method including administering to a subject in needthereof a first dose of any of the foregoing compounds or pharmaceuticalcompositions in an amount effective for radiation treatment planning,followed by administering subsequent doses of any of the foregoingcompounds or pharmaceutical compositions in a therapeutically effectiveamount.

In some embodiments, the compound or composition administered in thefirst dose and the compound or composition administered in the seconddose are the same.

In some embodiments, the compound or composition administered in thefirst dose and the compound or composition administered in the seconddose are different.

In some embodiments, the cancer is a solid tumor or hematologic (liquid)cancer.

In some embodiments, the solid tumor cancer is breast cancer, non-smallcell lung cancer, small cell lung cancer, pancreatic cancer, head andneck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocorticalcarcinoma, neuroendocrine cancer, Ewing's Sarcoma, multiple myeloma, oracute myeloid leukemia.

In some embodiments, the foregoing methods further include administeringan antiproliferative agent, radiation sensitizer, or an immunoregulatoryor immunomodulatory agent.

In some embodiments, any of the foregoing compounds or compositionsthereof and an antiproliferative agent or radiation sensitizer areadministered within 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1day(s)) of each other.

In some embodiments, any of the above-described compounds orcompositions thereof and an immunoregulatory or immunomodulatory agentare administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30,20, 10, 5, 4, 3, 2, or 1 day(s)) of each other.

In another aspect, the invention features a method of making aradioconjugate (e.g., any of the radioconjugates described herein). Themethod includes the steps of (a) conjugating a bifunctional chelate to abiological molecule, (b) purifying the conjugate produced by step (a),and (c) chelating one or more radionuclides (e.g., one or more Ac-225radionuclides) with the purified conjugate of step (b) at a temperatureof less than 35° C. (e.g., 20-25° C.) to produce a radioconjugate (e.g.an actinium radioconjugate).

In some embodiments, the radioconjugate is a radioimmunoconjugate (e.g.,any of the radioimmunoconjugates described herein).

In some embodiments, the pH of the reaction mixture of conjugation step(a) is less than 6.4 (e.g., 6.3, 6.2, 6.1, 6.0, 5.9, or 5.8 or less).

In some embodiments, the pH of the reaction mixture of conjugation step(c) is less than 5.5 (e.g., 5.4, 5.3, 5.2, 5.1, or 5.0 or less) or morethan 7.0 (e.g., 7.1, 7.2, 7.3, 7.4, 7.5 or more).

In some embodiments, the temperature of the reaction mixture ofconjugation step (c) is 20-34° C. (e.g., 21° C., 22° C., 23° C., 24° C.,25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C.,or 34° C.).

Chemical Terms:

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,trifluoroacetyl, propionyl, butanoyl and the like. Exemplaryunsubstituted acyl groups include from 1 to 7, from 1 to 11, or from 1to 21 carbons. In some embodiments, the alkyl group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxy, optionally substituted with an O-protecting group; (9) nitro;(10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇ spirocyclyl; (12)thioalkoxy; (13) thiol; (14) —CO₂R^(A)′, optionally substituted with anO-protecting group and where R^(A)′ is selected from the groupconsisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CHO_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B)′R^(C)′, where each of R^(B)′ and R^(C)′ is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D)′, where R^(D)′is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E)′R^(F)′, whereeach of R^(E)′ and R^(F)′ is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G)′, where R^(G)′ is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H)′C(O)R^(I)′, wherein R^(H)′ is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I)′ is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J)′C(O)OR^(K)′, wherein R^(J)′ is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K)′ is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionallysubstituted with an O-protecting group, such as optionally substitutedarylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl(e.g., acetyl, trifluoroacetyl, or others described herein),alkoxycarbonylalkyl (e.g., optionally substituted with an O-protectinggroup, such as optionally substituted arylalkoxycarbonyl groups or anydescribed herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl(e.g., alkheteroaryl), wherein each of these recited R^(N1) groups canbe optionally substituted, as defined herein for each group; or twoR^(N1) combine to form a heterocyclyl or an N-protecting group, andwherein each R^(N2) is, independently, H, alkyl, or aryl. The aminogroups of the invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others describedherein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl) or aryl, andeach R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A)′, where R^(A)′is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B)′R^(C)′, where each of R^(B)′ and R^(C)′ is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (C)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) -SO₂R^(D)′, where R^(D)′is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E)′R^(F)′, whereeach of R^(E)′ and R^(F)′ is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G)′, where R^(G)′ is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H)′C(O)R^(I)′, wherein R^(H)′ is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I)′ is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J)′C(O)OR^(K)′, wherein R^(J)′ is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K)′ is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A)′, where q is an integer from zero to four, andR^(A)′ is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B)′R^(C)′, where q is an integer from zero to four andwhere R^(B)′ and R^(C)′ are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D)′, where q is an integer fromzero to four and where R^(D)′ is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E)′R^(F)′, where q is an integer from zero to four andwhere each of R^(E)′ and R^(F)′ is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclylcan be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkyl,” as used herein, represents an aryl group, asdefined herein, attached to the parent molecular group through analkylene group, as defined herein.

Exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g.,from 7 to 16 or from 7 to 20 carbons, such as C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl). In some embodiments, thealkylene and the aryl each can be further substituted with 1, 2, 3, or 4substituent groups as defined herein for the respective groups. Othergroups preceded by the prefix “alk-” are defined in the same manner,where “alk” refers to a C₁₋₆ alkylene, unless otherwise noted, and theattached chemical structure is as defined herein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxy,” as used herein, means —CO₂H.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycle heptyl, andthe like. When the cycloalkyl group includes one carbon-carbon doublebond or one carbon-carbon triple bond, the cycloalkyl group can bereferred to as a “cycloalkenyl” or “cycloalkynyl” group respectively.Exemplary cycloalkenyl and cycloalkynyl groups include cyclopentenyl,cyclohexenyl, cyclohexynyl, and the like. The cycloalkyl groups of thisinvention can be optionally substituted with: (1) C₁₋₇ acyl (e.g.,carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A)′, where q isan integer from zero to four, and R^(A)′ is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B)′R^(C)′, where q is an integerfrom zero to four and where R^(B)′ and R^(C)′ are independently selectedfrom the group consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D)′, where q isan integer from zero to four and where R^(D)′ is selected from the groupconsisting of (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E)′R^(F)′, where q is an integer from zeroto four and where each of R^(E)′ and R^(F)′ is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halogen,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “heteroalkyl,” as used herein, refers to an alkyl group, asdefined herein, in which one or two of the constituent carbon atoms haveeach been replaced by nitrogen, oxygen, or sulfur. In some embodiments,the heteroalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups. The terms“heteroalkenyl” and heteroalkynyl,” as used herein refer to alkenyl andalkynyl groups, as defined herein, respectively, in which one or two ofthe constituent carbon atoms have each been replaced by nitrogen,oxygen, or sulfur. In some embodiments, the heteroalkenyl andheteroalkynyl groups can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heteroarylalkyl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein.

Exemplary unsubstituted heteroarylalkyl groups are from 2 to 32 carbons(e.g., from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to15, from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as C₁₋₆alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂ heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂heteroaryl). In some embodiments, the alkylene and the heteroaryl eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group. Heteroarylalkyl groups are asubset of heterocyclylalkyl groups.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H -purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H -purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂-, —NH—C(O)-, —NH—, —CH=N-,—CH₂—NH—, —C(O)-NH—, —CH=CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A)′, where q isan integer from zero to four, and R^(A)′ is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(a)R^(c)′, where q is an integerfrom zero to four and where R^(B)′ and R^(C)′ are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D)′, where q isan integer from zero to four and where R^(D)′ is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E)′R^(F)′, where q is an integer from zeroto four and where each of R^(E)′ and R^(F)′ is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxyl,” as used herein, represents an —OH group. In someembodiments, the hydroxyl group can be substituted with 1, 2, 3, or 4substituent groups (e.g., O-protecting groups) as defined herein for analkyl.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “O-protecting group,” as used herein, represents those groupsintended to protect an oxygen containing (e.g., phenol, hydroxyl, orcarbonyl) group against undesirable reactions during syntheticprocedures. Commonly used O-protecting groups are disclosed in Greene,“Protective Groups in Organic Synthesis,” 3^(rd) Edition (John Wiley &Sons, New York, 1999), which is incorporated herein by reference.Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups,such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl,tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl,phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and4-nitrobenzoyl; alkylcarbonyl groups, such as acyl, acetyl, propionyl,pivaloyl, and the like; optionally substituted arylcarbonyl groups, suchas benzoyl; silyl groups, such as trimethylsilyl (TMS),tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM),triisopropylsilyl (TIPS), and the like; ether-forming groups with thehydroxyl, such methyl, methoxymethyl, tetrahydropyranyl, benzyl,p-methoxybenzyl, trityl, and the like; alkoxycarbonyls, such asmethoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl,sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl,cyclohexyloxycarbonyl, methyloxycarbonyl, and the like;alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl,2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl,allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl,3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls, such as2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, and the like; optionally substitutedarylalkoxycarbonyl groups, such as benzyloxycarbonyl,p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl,3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxy-carbonyl, fluorenylmethyloxycarbonyl, and the like; andoptionally substituted aryloxycarbonyl groups, such as phenoxycarbonyl,p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl,2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl,m-methylphenoxycarbonyl, o-bromophenoxycarbonyl,3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl,2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl,aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl; methoxymethyl;benzyloxymethyl; siloxymethyl; 2,2,2,-trichloroethoxymethyl;tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl;1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether;p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl,and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; anddiphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl;vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl;and nitrobenzyl); carbonyl-protecting groups (e.g., acetal and ketalgroups, such as dimethyl acetal, 1,3-dioxolane, and the like; acylalgroups; and dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, andthe like); carboxylic acid-protecting groups (e.g., ester groups, suchas methyl ester, benzyl ester, t-butyl ester, orthoesters, and the like;and oxazoline groups.

The term “oxo” as used herein, represents ═O.

The term “polyethylene glycol,” as used herein, represents an alkoxychain comprised of one or more momomer units, each monomer unitconsisting of —OCH₂CH₂—. Polyethyelene glycol (PEG) is also sometimesreferred to as polyethylene oxide (PEO) or polyoxyethylene (POE), andthese terms may be considered interchangeable for the purpose of thisinvention. For example, a polyethylene glycol may have the structure,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)O—, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), and each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10). Polyethylene glycol mayalso be considered to include an amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1)_, wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thiol,” as used herein represents an —SH group.

Definitions

As used herein, the term “administered in combination” or “combinedadministration” means that two or more agents are administered to asubject at the same time or within an interval such that there may be anoverlap of an effect of each agent on the patient. In some embodiments,they are administered within 90 days (e.g., within 80, 70, 60, 50, 40,30, 20, 10, 5, 4, 3, 2, or 1 day(s)), within 28 days (e.g., with 14, 7,6, 5, 4, 3, 2, or 1 day(s), within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or1 hour(s), or within about 60, 30, 15, 10, 5, or 1 minute of oneanother. In some embodiments, the administrations of the agents arespaced sufficiently closely together such that a combinatorial (e.g., asynergistic) effect is achieved.

As used herein, “antibody” refers to a polypeptide whose amino acidsequence including immunoglobulins and fragments thereof whichspecifically bind to a designated antigen, or fragments thereof.Antibodies in accordance with the present invention may be of any type(e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1,IgG2, IgG3, or IgG4). Those of ordinary skill in the art will appreciatethat a characteristic sequence or portion of an antibody may includeamino acids found in one or more regions of an antibody (e.g., variableregion, hypervariable region, constant region, heavy chain, light chain,and combinations thereof). Moreover, those of ordinary skill in the artwill appreciate that a characteristic sequence or portion of an antibodymay include one or more polypeptide chains, and may include sequenceelements found in the same polypeptide chain or in different polypeptidechains.

As used herein, “antigen-binding fragment” refers to a portion of anantibody that retains the binding characteristics of the parentantibody.

The terms “bifunctional chelate” or “bifunctional conjugate” as usedinterchangeably herein, refer to a compound that contains a chelatinggroup or metal complex thereof, a linker group, and a therapeuticmoiety, targeting moiety, or cross linking group.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemia's, and lymphomas. A “solid tumor cancer” is a cancercomprising an abnormal mass of tissue, e.g., sarcomas, carcinomas, andlymphomas. A “hematological cancer” or “liquid cancer,” as usedinterchangeably herein, is a cancer present in a body fluid, e.g.,lymphomas and leukemias.

The term “chelate” as used herein, refers to an organic compound orportion thereof that can be bonded to a central metal or radiometal atomat two or more points.

The term “conjugate,” as used herein, refers to a molecule that containsa chelating group or metal complex thereof, a linker group, and whichoptionally contains a therapeutic moiety, targeting moiety, or crosslinking group.

As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, and tautomers of the structuresdepicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone—enol pairs, amide—imidicacid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl. Herein a phrase of the form “optionally substituted X”(e.g., optionally substituted alkyl) is intended to be equivalent to “X,wherein X is optionally substituted” (e.g., “alkyl, wherein said alkylis optionally substituted”). It is not intended to mean that the feature“X” (e.g. alkyl) per se is optional.

As used herein “detection agent” refers to a molecule or atom which isuseful in diagnosing a disease by locating the cells containing theantigen. Various methods of labeling polypeptides with detection agentsare known in the art. Examples of detection agents include, but are notlimited to, radioisotopes and radionuclides, dyes (such as with thebiotin-streptavidin complex), contrast agents, luminescent agents (e.g.,FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), andmagnetic agents, such as gadolinium chelates.

As used herein, the term “radionuclide,” refers to an atom capable ofundergoing radioactive decay (e.g. ³H, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁴⁷Sc, ⁵⁵Co,⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸⁹Zr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,⁹⁷Ru, ⁹⁹Tc, ^(99m)Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ₁₈₈Re, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰³Pb,²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, ^(229Th), ⁶⁶Ga, ⁶⁷Ga,⁶⁸Ga, ⁸²Rb, ^(117m)Sn, ²⁰¹TI). The terms radioactive nuclide,radioisotope, or radioactive isotope may also be used to describe aradionuclide. Radionuclides may be used as detection agents, asdescribed above. In some embodiments, the radionuclide may be analpha-emitting radionuclide.

The term an “effective amount” of an agent (e.g., any of the foregoingconjugates), as used herein, is that amount sufficient to effectbeneficial or desired results, such as clinical results, and, as such,an “effective amount” depends upon the context in which it is beingapplied.

The term “immunoconjugate,” as used herein, refers to a conjugate thatincludes a targeting moiety, such as an antibody, nanobody, affibody, ora consensus sequence from a Fibronectin type III domain. In someembodiments, the immunoconjugate comprises an average of at least 0.10conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 conjugates per targetingmoiety).

The term “radioconjugate,” as used herein, refers to any conjugate thatincludes a radioisotope or radionuclide, such as any of theradioisotopes or radionuclides described herein. In some embodiments theradioisotope or radionuclide is a metal chelate.

The term “radioimmunoconjugate,” as used herein, refers to anyimmunoconjugate that includes a radioisotope or radionuclide, such asany of the radioisotopes or radionuclides described herein. In someembodiments the radioisotope or radionuclide is a metal chelate.

The term “radioimmunotherapy,” as used herein, refers a method of usinga radioimmunoconjugate to produce a therapeutic effect. In someembodiments, radioimmunotherapy may include administration of aradioimmunoconjugate to a subject in need thereof, whereinadministration of the radioimmunoconjugate produces a therapeutic effectin the subject. In some embodiments, radioimmunotherapy may includeadministration of a radioimmunoconjugate to a cell, whereinadministration of the radioimmunoconjugate kills the cell. Whereinradioimmunotherapy involves the selective killing of a cell, in someembodiments the cell is a cancer cell in a subject having cancer.

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein formulated with apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is manufactured or sold with the approval ofa governmental regulatory agency as part of a therapeutic regimen forthe treatment of disease in a mammal. Pharmaceutical compositions can beformulated, for example, for oral administration in unit dosage form(e.g., a tablet, capsule, caplet, gelcap, or syrup); for topicaladministration (e.g., as a cream, gel, lotion, or ointment); forintravenous administration (e.g., as a sterile solution free ofparticulate emboli and in a solvent system suitable for intravenoususe); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, refers anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, radioprotectants, sorbents, suspending ordispersing agents, sweeteners, or waters of hydration. Exemplaryexcipients include, but are not limited to: ascorbic acid, histidine,phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc,titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts of the compounds described here that are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof humans and animals without undue toxicity, irritation, or allergicresponse. Pharmaceutically acceptable salts are well known in the art.For example, pharmaceutically acceptable salts are described in: Bergeet al., J. Pharmaceutical Sciences 66:1-19, 1977 and in PharmaceuticalSalts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G.Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during thefinal isolation and purification of the compounds described herein orseparately by reacting the free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be acid addition salts involving inorganic or organic acids or thesalts may, in the case of acidic forms of the compounds of the inventionbe prepared from inorganic or organic bases. Frequently, the compoundsare prepared or used as pharmaceutically acceptable salts prepared asaddition products of pharmaceutically acceptable acids or bases.Suitable pharmaceutically acceptable acids and bases are well-known inthe art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic,citric, or tartaric acids for forming acid addition salts, and potassiumhydroxide, sodium hydroxide, ammonium hydroxide, caffeine, variousamines for forming basic salts. Methods for preparation of theappropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,among others. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, and magnesium, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, andethylamine.

The term “therapeutic moiety” as used herein refers to any molecule orany part of a molecule that confers a therapeutic benefit. In someembodiments, the therapeutic moiety is a protein or polypeptide, e.g.,an antibody, an antigen-binding fragment thereof. In some embodiments,the therapeutic moiety is a small molecule.

The term “targeting moiety” as used herein refers to any molecule or anypart of a molecule that binds to a given target. In some embodiments,the targeting moiety is a protein or polypeptide such as an antibody orantigen binding fragment thereof, a nanobody, an affibody, or consensussequences from a Fibronectin type III domain.

The term “cross-linking group” as used herein refers to any reactivegroup that is able to join two or more molecules by a covalent bond. Insome embodiments, the cross-linking group is an amino-reactive orthiol-reactive cross-linking group. In some embodiments, theamino-reactive or thiol-reactive cross-linking group comprises anactivated ester such as a hydroxysuccinimide ester,2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate,anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne,strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide,diazirine, phosphine, tetrazine, isothiocyanate. In some embodiments,the cross linking group may by glycine-glycine-glycine and/orleucine-proline-(any amino acid)-threonine-glycine, which are therecognition sequences for coupling targeting agents with the linkerusing a sortase-mediated coupling reaction. The person having ordinaryskill in the art will understand that the use of cross linking groups inthe practice of the invention are not limited to the specific constructsdisclosed herein, but rather may include other known cross linkinggroups.

The term “polypeptide” as used herein refers to a string of at least twoamino acids attached to one another by a peptide bond. In someembodiments, a polypeptide may include at least 3-5 amino acids, each ofwhich is attached to others by way of at least one peptide bond. Thoseof ordinary skill in the art will appreciate that polypeptides caninclude one or more “non-natural” amino acids or other entities thatnonetheless are capable of integrating into a polypeptide chain. In someembodiments, a polypeptide may be glycosylated, e.g., a polypeptide maycontain one or more covalently linked sugar moieties. In someembodiments, a single “polypeptide” (e.g., an antibody polypeptide) maycomprise two or more individual polypeptide chains, which may in somecases be linked to one another, for example by one or more disulfidebonds or other means.

By “subject” is meant a human or non-human animal (e.g., a mammal).

By “substantial identity” or “substantially identical” is meant apolypeptide sequence that has the same polypeptide sequence,respectively, as a reference sequence, or has a specified percentage ofamino acid residues, respectively, that are the same at thecorresponding location within a reference sequence when the twosequences are optimally aligned. For example, an amino acid sequencethat is “substantially identical” to a reference sequence has at least50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the reference amino acid sequence. For polypeptides, thelength of comparison sequences will generally be at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150,200, 250, 300, or 350 contiguous amino acids (e.g., a full-lengthsequence). Sequence identity may be measured using sequence analysissoftware on the default setting (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Suchsoftware may match similar sequences by assigning degrees of homology tovarious substitutions, deletions, and other modifications.

As used herein, and as well understood in the art, “to treat” acondition or “treatment” of the condition (e.g., the conditionsdescribed herein such as cancer) is an approach for obtaining beneficialor desired results, such as clinical results. Beneficial or desiredresults can include, but are not limited to, alleviation or ameliorationof one or more symptoms or conditions; diminishment of extent ofdisease, disorder, or condition; stabilized (i.e., not worsening) stateof disease, disorder, or condition; preventing spread of disease,disorder, or condition; delay or slowing the progress of the disease,disorder, or condition; amelioration or palliation of the disease,disorder, or condition; and remission (whether partial or total),whether detectable or undetectable. “Palliating” a disease, disorder, orcondition means that the extent and/or undesirable clinicalmanifestations of the disease, disorder, or condition are lessenedand/or time course of the progression is slowed or lengthened, ascompared to the extent or time course in the absence of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the general structure of a conjugatecomprising a chelate, a linker, and a cross-linking group (top) and aconjugate comprising a chelate, a linker, and a targeting moiety(bottom).

FIG. 2 is a schematic depicting the synthesis of the bifunctionalchelate,4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoicacid (Compound B). The synthesis of Compound B is described in Example3.

FIG. 3 is a schematic depicting the synthesis of the bifunctionalchelate,4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoicacid (Compound C). The synthesis of Compound C is described in Example4.

FIG. 4 is a series of graphs depicting the metabolic excretion profileof non-targeted human IgG antibody conjugates [¹⁷⁷Lu]-Compound B-HuMIgG,and [¹⁷⁷Lu]-Compound C-HuMIgG as compared to [¹⁷⁷Lu]-Compound A-HuMIgG,the methods and results of which are described in detail in Example 7.

DETAILED DESCRIPTION

Radiolabelled targeting moieties (also known as radioimmunoconjugates)are designed to target a protein or receptor that is upregulated in adisease state to deliver a radioactive payload to kill cells of interest(radioimmunotherapy). The process of delivering such a payload, viaradioactive decay, produces the emission of an alpha, beta, or gammaparticle or Auger electron that can cause direct effects to DNA (such assingle or double stranded DNA breaks) or indirect effects such asby-stander or crossfire effects.

Radioimmunoconjugates typically contain a biological targeting moiety, aradioisotope, and a conjugate that links the two. Conjugates are formedwhen a bifunctional chelate is appended to the biological targetingmolecule so that structural alterations to the compound are minimalwhile maintaining target affinity. Once radiolabelled, the finalradioimmunoconjugate is formed.

Bifunctional chelates structurally contain a chelate, the linker, andcross-linking group (FIG. 1). When developing new bifunctional chelates,most efforts focus around the chelating portion of the molecule. Severalexamples of bifunctional chelates have been described with variouscyclic and acyclic structures that when conjugated to a targeted moiety.[Bioconjugate Chem. 2000, 11, 510-519, Bioconjugate Chem.2012, 23,1029-1039, Mol Imaging Biol (2011) 13:215-221, BioconjugateChem.2002,13,110-115]

One the key factors of developing safe and effectiveradioimmunoconjugates is maximizing efficacy while minimizing off-targettoxicity in normal tissue. While this statement is one of the coretenants of developing new drugs, the application toradioimmunotherapeutics presents new challenges. Radioimmunoconjugatesdo not need to block a receptor, as needed with a therapeutic antibody,or release the cytotoxic payload intracellularly, as required with anantibody drug conjugate, in order to have therapeutic efficacy. However,emission of the toxic particle is an event that occurs as a result offirst-order (radioactive) decay and can occur at random anywhere insidethe body after administration. Once the particle is released, damagecould occur to surrounding cells within the range of the emissionleading to the potential of off-target toxicity. Therefore, limitingexposure of these emissions to normal tissue is the key to developingnew drugs [Bioconjugate Chem. 2006, 17, 1551-1560, Bioconjugate Chem.2003, 14, 927-933].

One method potential method for reducing off-target exposure is toremove the radioactivity more effectively from the body. The mostobvious mechanism is increase the rate of clearance of the biologicaltargeting agent. This approach also likely requires identifying ways toshorten the half-life of the biological, which is a topic not welldescribed for biological targeting agents. Regardless of the mechanism,increasing drug clearance will also negatively impact thepharmacodynamics/efficacy in that the more rapid removal of drug fromthe body will lower the effective concentration at the site of action,which, in turn, would require a higher total dose and would not achievethe desired results of reducing total radioactive dose to normal tissue.

Other efforts have focused on accelerating the metabolism of the portionof the molecule that contains the radioactive moiety. To this end, someefforts have been made to increase the rate of cleavage of theradioactivity from the biological targeting agents using what have beentermed “cleavable linkers”. Cleavable linkers, however, have been takenon different meaning as it relates to radioimmunoconjugates.Cornelissen, et al. has described cleavable linkers as those by whichthe bifunctional conjugate attaches to the biologic targeting agentthrough a reduced cysteine, whereas others have described the use ofenzyme-cleavable systems that require the co-administration of theradioimmunoconjugate with a cleaving agent/enzyme to release [Mol CancerTher; 12(11) November 2013, Methods in Molecular Biology, 2009, 539,191-211, Bioconjugate chemistry, Volume 14, Issue 5, p.927-33 (2003)].These methods either change the nature of the biological targetingmoiety, in the case of the cysteine linkage, or are not practical from adrug development perspective (enzyme cleavable systems) since, in thecase of the citations provided, these methods require the administrationof 2 agents.

The focus of the embodiments described herein centers on moreeffectively eliminating radioactivity from the body after catabolismand/or metabolism of the radioimmunoconjugate by making modifications tothe linker region of the bifunctional chelate.

This is a novel approach since little information appears to existdescribing the in vivo impact of the linker, especially as it applies toradioimmunoconjugates. One potential reason is that followingcatabolism/metabolism of the radioimmunoconjugate, one would expect theradiolabelled conjugate to undergo rapid systemic elimination. Thesupposition was furthered experimentally when the bifunctional chelatewas administered alone; it cleared the bloodstream faster than theradioimmunoconjugate with that same bifunctional chelate. Based on thisdata, one would expect that following catabolism/metabolism of theradioimmunoconjugate, the metabolite containing the bifunctional chelatewould also be rapidly eliminated.

However, rapid clearance of the metabolites containing the radiolabelledconjugate does not necessarily occur in vivo. Based on the resultsdescribed below, the linker region of bifunctional chelates can directlyimpact the elimination of the radioactivity from the body followingcatabolism of the radioconjugate while not impacting the overall invitro properties or the in vivo pharmacokinetics and pharmacodynamics ofthe radioimmunoconjugate. Data is presented below that demonstrates thatthe certain bifunctional chelates available commercially produce aslower rate and a lower extent of elimination of the total radioactivityfrom the body when compared to the embodiments described herein.

The excretion profiles of the embodiments described in the Examplesrepresent unexpected findings. As previously reported, Quadri andVriesendorp [Q. J. Nucl. Med. 1998, 42, 250-261], simple modificationsto the linker region of the bifunctional chelate, regardless of theirhydrophobicity, did not impact urinary excretion of the radioactivity.The results provided below clearly indicate that both hydrophobic andhydrophilic linkers can impact excretion patterns. In addition, theExamples below demonstrate that hepatobiliary clearance also plays arole in excretion.

Therefore, through the embodiments described herein, bifunctionalchelates, when attached to biological targeting moieties, have beenidentified that achieve a reduction of total body radioactivity byincreasing the extent of excretion of the catabolic/metabolic productswhile maintaining the pharmacokinetics of the intact molecule whencompared to similar bifunctional chelates in the public domain. Thisreduction in total body radioactivity has been determined to be due tothe clearance of catabolic/metabolic by-products and does not impact theother in vitro and in vivo properties such as degree of specificity (invitro binding), cellular retention, and tumor uptake in vivo. When takenin whole, these embodiments achieve the desired properties ofradioimmunoconjugates by reducing the body burden of radioactivity whilemaintaining on-target activity.

Therapeutic Moieties and Targeting Moieties

Therapeutic moieties include any molecule or any part of a molecule thatconfers a therapeutic benefit. In some embodiments, the therapeuticmoiety is a protein or polypeptide, e.g., an antibody, anantigen-binding fragment thereof. In some embodiments, the therapeuticmoiety is a small molecule. Targeting moieties include any molecule orany part of a molecule that binds to a given target. In someembodiments, the targeting moiety is a protein or polypeptide such asantibodies or antigen binding fragments thereof, nanobodies, affibodies,and consensus sequences from Fibronectin type III domains (e.g.,Centyrins or Adnectins).

Polypeptides

Polypeptides include, for example, any of a variety of hematologicagents (including, for instance, erythropoietin, blood-clotting factors,etc.), interferons, colony stimulating factors, antibodies, enzymes, andhormones. The identity of a particular polypeptide is not intended tolimit the present disclosure, and any polypeptide of interest can be apolypeptide in the present methods.

A reference polypeptide described herein can include a target-bindingdomain that binds to a target of interest (e.g., binds to an antigen).For example, a polypeptide, such as an antibody, can bind to atransmembrane polypeptide (e.g., receptor) or ligand (e.g., a growthfactor). Exemplary molecular targets (e.g., antigens) for polypeptidesdescribed herein (e.g., antibodies) include CD proteins such as CD2,CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52;members of the ErbB receptor family such as the EGF receptor (EGFR,HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor;macrophage receptors such as CRIg; tumor necrosis factors such as TNFαor TRAIL/Apo-2; cell adhesion molecules such as LFA-1, Mac1, p150,95,VLA-4, ICAM-1, VCAM and avβ3 integrin including either α or β subunitsthereof (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growthfactors and receptors such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE;cytokines such as IL1; cytokine receptors such as IL2 receptor; bloodgroup antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;CTLA-4; protein C; neutropilins; ephrins and receptors; netrins andreceptors; slit and receptors; chemokines and chemokine receptors suchas CCL5, CCR4, CCR5; amyloid beta; complement factors, such ascomplement factor D; lipoproteins, such as oxidized LDL (oxLDL);lymphotoxins, such as lymphotoxin alpha (LTa). Other molecular targetsinclude Tweak, B7RP-1, proprotein convertase subtilisin/kexin type 9(PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

Antibodies

An IgG antibody consists of two identical light polypeptide chains andtwo identical heavy polypeptide chains linked together by disulfidebonds. The first domain located at the amino terminus of each chain isvariable in amino acid sequence, providing the antibody bindingspecificities found in each individual antibody. These are known asvariable heavy (VH) and variable light (VL) regions. The other domainsof each chain are relatively invariant in amino acid sequence and areknown as constant heavy (CH) and constant light (CL) regions. For an IgGantibody, the light chain includes one variable region (VL) and oneconstant region (CL). An IgG heavy chain includes a variable region(VH), a first constant region (CH1), a hinge region, a second constantregion (CH2), and a third constant region (CH3). In IgE and IgMantibodies, the heavy chain includes an additional constant region(CH4).

Antibodies described herein can include, for example, monoclonalantibodies, polyclonal antibodies, multispecific antibodies, humanantibodies, humanized antibodies, camelid antibodies, chimericantibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), andanti-idiotypic (anti-Id) antibodies, and antigen-binding fragments ofany of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass.

The term “antigen binding fragment” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen. Examples of binding fragmentsencompassed within the term “antigen binding fragment” of an antibodyinclude a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fvfragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature341:544-546), and an isolated complementarity determining region (CDR).These antibody fragments can be obtained using conventional techniquesknown to those with skill in the art, and the fragments can be screenedfor utility in the same manner as are intact antibodies.

Antibodies or fragments described herein can be produced by any methodknown in the art for the synthesis of antibodies (see, e.g., Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can beproduced using the methods described in, e.g., Morrison, 1985, Science229:1202, and humanized antibodies by methods described in, e.g., U.S.Pat. No. 6,180,370.

Additional antibodies described herein are bispecific antibodies andmultivalent antibodies, as described in, e.g., Segal et al., J. Immunol.Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).

Insulin-Like Growth Factor 1 (IGF-1R) Antibodies

Insulin-like growth factor 1 receptor is a transmembrane protein foundon the surface of human cells activated by insulin-like growth factor 1(IGF-1) and 2 (IGF-2). IGF-1R is implicated in several cancers includingbreast cancer, non-small cell lung cancer, prostate cancer, coloncancer, sarcoma, and adrenocortical carcinoma, increased levels ofIGF-1R are expressed on the surface of tumor cells of these cancers.

In some embodiments, the antibody, or an antigen-binding fragmentthereof specifically binds insulin-like growth factor-1 receptor(IGF-1R).

Nanobodies

Nanobodies are antibody fragments consisting of a single monomericvariable antibody domain. Nanobodies may also be referred to assingle-domain antibodies. Like antibodies, nanobodies bind selectivelyto a specific antigen. Nanobodies may be heavy-chain variable domains orlight chain domains. Nanobodies may occur naturally or be the product ofbiological engineering. Nanobodies may be biologically engineered bysite-directed mutagenesis or mutagenic screening (e.g., phage display,yeast display, bacterial display, mRNA display, ribosome display).

Affibodies

Affibodies are polypeptides or proteins engineered to bind to a specificantigen. As such, affibodies may be considered to mimic certainfunctions of antibodies. Affibodies may be engineered variants of theB-domain in the immunoglobulin-binding region of staphylococcal proteinA. Affibodies may be engineered variants of the Z-domain, a B-domainthat has lower affinity for the Fab region. Affibodies may bebiologically engineered by site-directed mutagenesis or mutagenicscreening (e.g., phage display, yeast display, bacterial display, mRNAdisplay, ribosome display).

Affibody molecules showing specific binding to a variety of differentproteins (e.g. insulin, fibrinogen, transferrin, tumor necrosisfactor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2and EGFR) have been generated, demonstrating affinities (K_(d)) in thepM to pM range.

Fibronectin Type III Domains

The Fibronectin type III domain is an evolutionarily conserved proteindomain found in a wide-variety of extracellular proteins. TheFibronectin type III domain has been used as a molecular scaffold toproduce molecules capable of selectively binding a specific antigen.Variants of the Fibronectin type III domains (FN3) that have beenengineered for selective-binding may also be referred to as monobodies.FN3 domains may be biologically engineered by site-directed mutagenesisor mutagenic screening (e.g., CIS-display, phage display, yeast display,bacterial display, mRNA display, ribosome display).

Modified Polypeptides

The polypeptides of the invention may have a modified amino acidsequence. Modified polypeptides may be substantially identical to thecorresponding reference polypeptide (e.g., the amino acid sequence ofthe modified polypeptide may have at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acidsequence of the reference polypeptide). In certain embodiments, themodification does not destroy significantly a desired biologicalactivity. The modification may reduce (e.g., by at least 5%, 10%, 20%,25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, ormay increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or1000%) the biological activity of the original polypeptide. The modifiedpolypeptide may have or may optimize a characteristic of a polypeptide,such as in vivo stability, bioavailability, toxicity, immunologicalactivity, immunological identity, and conjugation properties.

Modifications include those by natural processes, such aspost-translational processing, or by chemical modification techniquesknown in the art. Modifications may occur anywhere in a polypeptideincluding the polypeptide backbone, the amino acid side chains and theamino- or carboxy-terminus. The same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide,and a polypeptide may contain more than one type of modification.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched, and branchedcyclic polypeptides may result from post-translational natural processesor may be made synthetically. Other modifications include pegylation,acetylation, acylation, addition of acetomidomethyl (Acm) group,ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation,carboxyethylation, esterification, covalent attachment to fiavin,covalent attachment to a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of drug,covalent attachment of a marker (e.g., fluorescent or radioactive),covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent crosslinks, formation ofcystine, formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation and ubiquitination.

A modified polypeptide can also include an amino acid insertion,deletion, or substitution, either conservative or non-conservative(e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g.,where such changes do not substantially alter the biological activity ofthe polypeptide). In particular, the addition of one or more cysteineresidues to the amino or carboxy terminus of any of the polypeptides ofthe invention can facilitate conjugation of these polypeptides by, e.g.,disulfide bonding. For example, a polypeptide can be modified to includea single cysteine residue at the amino-terminus or a single cysteineresidue at the carboxy-terminus. Amino acid substitutions can beconservative (i.e., wherein a residue is replaced by another of the samegeneral type or group) or non-conservative (i.e., wherein a residue isreplaced by an amino acid of another type). In addition, a naturallyoccurring amino acid can be substituted for a non-naturally occurringamino acid (i.e., non-naturally occurring conservative amino acidsubstitution or a non-naturally occurring non-conservative amino acidsubstitution).

Polypeptides made synthetically can include substitutions of amino acidsnot naturally encoded by DNA (e.g., non-naturally occurring or unnaturalamino acid). Examples of non-naturally occurring amino acids includeD-amino acids, N-protected amino acids, an amino acid having anacetylaminomethyl group attached to a sulfur atom of a cysteine, apegylated amino acid, the omega amino acids of the formulaNH₂(CH₂)_(n)COOH wherein n is 2-6, neutral nonpolar amino acids, such assarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, andnorleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;citrulline and methionine sulfoxide are neutral nonpolar, cysteic acidis acidic, and ornithine is basic. Proline may be substituted withhydroxyproline and retain the conformation conferring properties.

Analogs may be generated by substitutional mutagenesis and retain thebiological activity of the original polypeptide. Examples ofsubstitutions identified as “conservative substitutions” are shown inTable 1. If such substitutions result in a change not desired, thenother type of substitutions, denominated “exemplary substitutions” inTable 1, or as further described herein in reference to amino acidclasses, are introduced and the products screened.

TABLE 1 Amino acid substitutions Original Conservative residue Exemplarysubstitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gln, Lys,Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, SerPhe Val (V) Ile, Leu, Met, Phe, Ala, Leu norleucine

Substantial modifications in function or immunological identity areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Cross-Linking Groups

A cross-linking group is a reactive group that is able to join two ormore molecules by a covalent bond. Cross-linking groups may be used toattach the linker and chelating moiety to a therapeutic or targetingmoiety. Cross-linking groups may also be used to attach the linker andchelating moiety to a target in vivo. In some embodiments, thecross-linking group is an amino-reactive or thiol-reactive cross-linkinggroup, or a sortase-mediated coupling. In some embodiments, theamino-reactive, methionine reactive or thiol-reactive cross-linkinggroup comprises an activated ester such as a hydroxysuccinimide ester,2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate,anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne,strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide,diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine. In someembodiments, the sortase recognition sequence may comprise of a terminalglycine-glycine-glycine (GGG) and/or LPTXG amino acid sequence, where Xis any amino acid. The person having ordinary skill in the art willunderstand that the use of cross linking groups in the practice of theinvention are not limited to the specific constructs disclosed herein,but rather may include other known cross linking groups.

Detection Agents

A detection agent is a molecule or atom which is administered conjugatedto a polypeptide, e.g., an antibody or antigen-binding fragment thereof,and is useful in diagnosing a disease by locating the cells containingthe antigen, radiation treatment planning, or treatment of a disease.Useful detection agents include, but are not limited to, radioisotopes,dyes (such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules, luminescent agents, and enhancingagents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).In order to load a polypeptide component with a detection agent it maybe necessary to react it with a reagent having a linker to which areattached the detection agent or multiple detection agents.

Radioisotopes and Radionuclides

Radioisotopes and radionuclides known in the art for their utility asdetection agents include, but are not limited to, ³H, ¹⁴C, ¹⁵N, ¹⁸F,³⁵S, ⁴⁷Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸⁹Zr,⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ^(99m)Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ₁₈₈Re, ¹⁹⁸Au,¹⁹⁹Au, ²⁰³Pb, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, ^(229Th),⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ^(117m)Sn, ²⁰¹TI.

Chelating Moieties

Chelating moieties are known in the art for their utility as detectionagents include, but are not limited to, DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α, α′, α″,α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid),DO3AM-acetic acid(2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)aceticacid), DOTA-GA anhydride(2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-tril)triaceticacid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP(1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid,DOTA-4AMP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonicacid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP(1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA(1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid),PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaaceticacid), H₄octapa(N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diaceticacid), H₂dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane),H₆phospa(N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane),TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P(tetraazacyclododecane dimethanephosphonic acid), HP-DO3A(hydroxypropyltetraazacyclododecanetriacetic acid), EDTA(ethylenediaminetetraacetic acid), Deferoxamine, DTPA(diethylenetriaminepentaacetic acid), DTPA-BMA(diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentatehydroxypyridinones), or porphyrins. Chelating groups may be used inmetal chelate combinations with metals, such as manganese, iron, andgadolinium and isotopes (e.g., isotopes in the general energy range of60 to 4,000 keV), such as ₄₇Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁸Ga, ⁸²Rb, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁹⁷Ru, ^(99m)Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In,^(117m)Sn, ¹⁴⁹Tb, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²⁰¹TI,²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th.

Linkers

Linkers of the invention may have the structure of Formula I:

A-L¹-(L²)_(n)-B   Formula I

wherein A is chelating moiety or a metal complex thereof;

-   L¹ is optionally substituted C₁-C₆ alkyl, substituted C₁-C₆    heteroalkyl, substituted aryl or heteroaryl;-   B is a is a therapeutic moiety, a targeting moiety, or cross-linking    group,-   or a pharmaceutically acceptable salt thereof;-   n is 1-5;-   each L², independently, has the structure:

(—X¹-L³-Z¹—)   Formula II

wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹),—CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹) , O, NR¹ and R¹ is H or optionallysubstituted C₁-C₆ alkyl or optionally substituted C₁-C₆ heteroalkyl,substituted aryl or heteroaryl;

-   L³ is optionally substituted C₁-C₅₀ alkyl or optionally substituted    C₁-C₅₀ heteroalkyl or C₅₋₂₀ polyethylene glycol; Z¹ is CH₂, C═O,    C═S, OC═O, NR¹C═O, NR¹ and R¹ is a hydrogen or optionally    substituted C₁₋₆ alkyl, pyrrolidine-2,5-dione.

The conjugates of the invention comprise three distinct modules thattogether result in their increased effectiveness compared to those knownin the art.

1. Chelating Moiety or Metal Complex Thereof:

-   Module A is included for incorporation of a detection agent (e.g., a    chelating moiety or metal complex thereof). A metal complex may    include an imaging radionuclide.

2. Linkers:

-   Linkers of the invention have the structure of Formula I:

A-L¹-(L²)_(n)-B   Formula I

wherein A is chelating moiety or a metal complex thereof;

L¹ is optionally substituted C₁-C₆ alkyl, substituted C₁-C₆ heteroalkyl,substituted aryl or heteroaryl;

B is a is a therapeutic moiety, a targeting moiety, or cross-linkinggroup,

or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L², independently, has the structure:

(—X¹-L³-Z¹—)   Formula II

wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹),—CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹), O, NR¹ and R¹ is H or optionallysubstituted C₁-C₆ alkyl or optionally substituted C₁-C₆ heteroalkyl,substituted aryl or heteroaryl;

L³ is optionally substituted C₁-C₅₀ alkyl or optionally substitutedC₁-C₅₀ heteroalkyl or C₅-C₂₀ polyethylene glycol;

Z¹ is CH₂, C═O, C═S, OC═O, NR¹C═O, NR¹ and R¹ is a hydrogen oroptionally substituted C₁-C₆ alkyl, pyrrolidine-2,5-dione.

3. Therapeutic Moiety, Targeting Moiety, or Cross-Linking Group:

-   Module B is a therapeutic moiety (e.g., antibodies, antigen-binding    fragments), a targeting moiety (e.g. nanobodies, affibodies,    consensus sequences from Fibronectin type Ill domains), or a    cross-linking group (e.g. amino-reactive, thiol-reactive    cross-linking group, or a sortase-mediated coupling).

Administration and Dosage

The present invention also features pharmaceutical compositions thatcontain a therapeutically effective amount of a compound of theinvention. The composition can be formulated for use in a variety ofdrug delivery systems. One or more physiologically acceptable excipientsor carriers can also be included in the composition for properformulation. Suitable formulations for use in the present invention arefound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985. For a brief review of methods fordrug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

The pharmaceutical compositions are intended for parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for prophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered parenterally (e.g., by intravenous,intramuscular, or subcutaneous injection), or by oral ingestion, or bytopical application or intraarticular injection at areas affected by thevascular or cancer condition. Additional routes of administrationinclude intravascular, intra-arterial, intratumor, intraperitoneal,intraventricular, intraepidural, as well as nasal, ophthalmic,intrascleral, intraorbital, rectal, topical, or aerosol inhalationadministration. Sustained release administration is also specificallyincluded in the invention, by such means as depot injections or erodibleimplants or components. Thus, the invention provides compositions forparenteral administration that include the above mention agentsdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, or PBS, among others. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, or detergents, among others. The invention also providescompositions for oral delivery, which may contain inert ingredients suchas binders or fillers for the formulation of a unit dosage form, such asa tablet or a capsule. Furthermore, this invention provides compositionsfor local administration, which may contain inert ingredients such assolvents or emulsifiers for the formulation of a cream, an ointment, agel, a paste, or an eye drop.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 6 and 7, such as 6 to 6.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The compositions containing an effective amount can be administered forradiation treatment planning, diagnostic, or therapeutic treatments.When administered for radiation treatment planning or diagnosticpurposes, the conjugate is administered to a subject in a diagnosticallyeffective dose and/or an amount effective to determine thetherapeutically effective dose. In therapeutic applications,compositions are administered to a subject (e.g., a human) alreadysuffering from a condition (e.g., cancer) in an amount sufficient tocure or at least partially arrest the symptoms of the disorder and itscomplications. An amount adequate to accomplish this purpose is definedas a “therapeutically effective amount,” an amount of a compoundsufficient to substantially improve at least one symptom associated withthe disease or a medical condition. For example, in the treatment ofcancer, an agent or compound that decreases, prevents, delays,suppresses, or arrests any symptom of the disease or condition would betherapeutically effective. A therapeutically effective amount of anagent or compound is not required to cure a disease or condition butwill provide a treatment for a disease or condition such that the onsetof the disease or condition is delayed, hindered, or prevented, or thedisease or condition symptoms are ameliorated, or the term of thedisease or condition is changed or, for example, is less severe orrecovery is accelerated in an individual. The conjugates of theinvention can be used for the treatment of cancer by administering to asubject a first dose of any of the foregoing conjugates or compositionsin an amount effective for radiation treatment planning, followed byadministering a second dose of any of the foregoing conjugates orcompositions in a therapeutically effective amount.

Amounts effective for these uses may depend on the severity of thedisease or condition and the weight and general state of the subject.The therapeutically effective amount of the compositions of theinvention and used in the methods of this invention applied to mammals(e.g., humans) can be determined by the ordinarily-skilled artisan withconsideration of individual differences in age, weight, and thecondition of the mammal. Because certain conjugates of the inventionexhibit an enhanced ability to target cancer cells and residualize, thedosage of the compounds of the invention can be lower than (e.g., lessthan or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose ofrequired for a therapeutic effect of the unconjugated agent. The agentsof the invention are administered to a subject (e.g., a mammal, such asa human) in an effective amount, which is an amount that produces adesirable result in a treated subject. Therapeutically effective amountscan also be determined empirically by those of skill in the art.

Single or multiple administrations of the compositions of the inventionincluding an effective amount can be carried out with dose levels andpattern being selected by the treating physician. The dose andadministration schedule can be determined and adjusted based on theseverity of the disease or condition in the subject, which may bemonitored throughout the course of treatment according to the methodscommonly practiced by clinicians or those described herein.

The conjugates of the present invention may be used in combination witheither conventional methods of treatment or therapy or may be usedseparately from conventional methods of treatment or therapy.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a compound of the present invention in association with apharmaceutically acceptable excipient, as described herein, and anothertherapeutic or prophylactic agent known in the art.

By “antiproliferative” or “antiproliferative agent,” as usedinterchangeably herein, is meant any anticancer agent, including thoseantiproliferative agents listed in Table 2, any of which can be used incombination with a conjugate of the invention to treat the medicalconditions recited herein. Antiproliferative agents also includeorgano-platinum derivatives, naphtoquinone and benzoquinone derivatives,chrysophanic acid and anthroquinone derivatives thereof.

By “immunoregulatory agent” or “immunomodulatory agent,” as usedinterchangeably herein, is meant any immuno-modulator, including thoselisted in Table 2, any of which can be used in combination with aconjugate of the invention to treat the medical conditions recitedherein.

As used herein, “radiation sensitizer” includes any agent that increasesthe sensitivity of cancer cells to radiation therapy. Radiationsensitizers may include, but are not limited to, 5′fluorouracil, analogsof platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine,EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferaseinhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF antagonists.

TABLE 2 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazineifosfamide altretamine hexamethylmelamine estramustine phosphatethiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomidecyclophosphamide Semustine Platinum agents spiroplatin lobaplatin(Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464(Hoffmann-La Roche) iproplatin SM-11355 (Sumitomo) picoplatin AP-5280(Access) oxaliplatin cisplatin carboplatin Antimetabolites azacytidinetrimetrexate Floxuridine deoxycoformycin 2-chlorodeoxyadenosinepentostatin 6-mercaptopurine hydroxyurea 6-thioguanine decitabine(SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidineirofulven (MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) tomudexethynylcytidine (Taiho) fludarabine gemcitabine raltitrexed capecitabineTopoisomerase amsacrine exatecan mesylate (Daiichi) inhibitorsepirubicin quinamed (ChemGenex) etoposide gimatecan (Sigma-Tau)teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen)7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) dexrazoxanet(TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) J-107088(Merck & Co) rebeccamycin analogue (Exelixis) BNP-1350 (BioNumerik)BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang) rubitecan (SuperGen)KW-2170 (Kyowa Hakko) irinotecan (CPT-11) hydroxycamptothecin (SN-38)topotecan Antitumor antibiotics valrubicin azonafide therarubicinanthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycinMEN-10755 (Menarini) porfiromycin GPX-100 (Gem Pharmaceuticals)mitoxantrone (novantrone) Epirubicin amonafide mitoxantrone doxorubicinAntimitotic colchicine E7010 (Abbott) agents vinblastine PG-TXL (CellTherapeutics) vindesine IDN 5109 (Bayer) dolastatin 10 (NCl) A 105972(Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin(Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 (ASTAMedica)RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatinA4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar) T900607 (Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ10992(Asahi) cryptophycin 52 (Eli Lilly) IDN-5109 (Indena) vinflunine (Fabre)AVLB (Prescient NeuroPharma) auristatin PE (Teikoku Hormone)azaepothilone B (BMS) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476(BMS) CA-4 prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH)taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075 (GlaxoSmithKline)docetaxel Vinorelbine vincristine Trichostatin A paclitaxel Aromataseinhibitors aminoglutethimide YM-511 (Yamanouchi) atamestane(BioMedicines) formestane letrozole exemestane anastrazole Thymidylatepemetrexed (Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331(BTG) CoFactor ™ (BioKeys) DNA antagonists trabectedin (PharmaMar)edotreotide (Novartis) glufosfamide (Baxter International) mafosfamide(Baxter International) albumin + 32P (Isotope Solutions) apaziquone(Spectrum thymectacin (NewBiotics) Pharmaceuticals) O6 benzyl guanine(Paligent) Farnesyltransferase arglabin (NuOncology Labs) tipifarnib(Johnson & Johnson) inhibitors lonafarnib (Schering-Plough) perillylalcohol (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBAPharma) zosuquidar trihydrochloride (Eli Lilly) tariquidar (Xenova)biricodar dicitrate (Vertex) MS-209 (Schering AG) Histone tacedinaline(Pfizer) pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA (AtonPharma) depsipeptide (Fujisawa) inhibitors MS-275 (Schering AG)Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex)inhibitors marimastat (British Biotech) BMS-275291 (Celltech)Ribonucleoside gallium maltolate (Titan) tezacitabine (Aventis)reductase inhibitors triapine (Vion) didox (Molecules for Health) TNFalpha virulizin (Lorus Therapeutics) revimid (Celgene)agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan (Abbott)YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoicacid fenretinide (Johnson & Johnson) alitretinoin (Ligand) receptoragonists LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy(Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK(Progenics) Technology) adenocarcinoma vaccine (Biomira) ISF-154(Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2(Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira)synchrovax vaccines (CTL Immuno) MGV (Progenics) melanoma vaccine (CTLImmuno) β-alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy(Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCamBiotherapeutics) abatacept (BMS) atezolizumab (Genentech) pembrolizumab(Merck) Hormonal and estrogens dexamethasone antihormonal agentsconjugated estrogens prednisone ethinyl estradiol methylprednisolonechlortrianisen prednisolone idenestrol aminoglutethimidehydroxyprogesterone caproate leuprolide medroxyprogesterone octreotidetestosterone mitotane testosterone propionate; P-04 (Novogen)fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosteronearzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofinebicalutamide goserelin flutamide Leuporelin nilutamide bicalutamidePhotodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda)agents Theralux (Theratechnologies) lutetium texaphyrin (Pharmacyclics)motexafin gadolinium hypericin (Pharmacyclics) Kinase Inhibitorsimatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia)kahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib(Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518(Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia)Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190(AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210(Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447(Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth)IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774(Tarceva ™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis)SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) EmodinGenistein Radicinol Radicinol Vemurafenib (B-Raf enzyme Met-MAb (Roche)inhibitor, Daiichi Sankyo) SR-27897 (CCK A inhibitor, Sanofi-Synthelabo)ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMPagonist, Ribapharm) BOX-1777 (PNP inhibitor, BioCryst) alvocidib (CDKinhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247(COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor,Dong-A) P54 (COX-2 inhibitor, Phytopharm) tirapazamine (reducing agent,SRI CapCell ™ (CYP450 stimulant, Bavarian Nordic) International) GCS-100(gal3 antagonist, GlycoGenesys) N-acetylcysteine (reducing agent,Zambon) G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen(NF-kappaB inhibitor, Encore) efaproxiral (oxygenator, AllosTherapeutics) 3CPA (NF-kappaB inhibitor, Active Biotech) PI-88(heparanase inhibitor, Progen) seocalcitol (vitamin D receptor agonist,Leo) tesmilifene (histamine antagonist, YM 131-I-TM-601 (DNA antagonist,BioSciences) TransMolecular) histamine (histamine H2 receptor agonist,Maxim) eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDHinhibitor, Ribapharm) minodronic acid (osteoclast inhibitor, cilengitide(integrin antagonist, Merck KGaA) Yamanouchi) SR-31747 (IL-1 antagonist,Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinaseinhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE Vinhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst)CP-461 (PDE V inhibitor, Cell Pathways) PG2 (hematopoiesis enhancer,AG-2037 (GART inhibitor, Pfizer) Pharmagenesis) WX-UK1 (plasminogenactivator inhibitor, Wilex) Immunol ™ (triclosan oral rinse, Endo)PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine(uridine prodrug, Wellstat) bortezomib (proteasome inhibitor,Millennium) SN-4071 (sarcoma agent, Signature SRL-172 (T cell stimulant,SR Pharma) BioScience) TLK-286 (glutathione S transferase inhibitor,TransMID-107 ™ (immunotoxin, KS Biomedix) Telik) PCK-3145 (apoptosispromotor, Procyon) PT-100 (growth factor agonist, Point doranidazole(apoptosis promotor, Pola) Therapeutics) CHS-828 (cytotoxic agent, Leo)midostaurin (PKC inhibitor, Novartis) trans-retinoic acid(differentiator, NIH) bryostatin-1 (PKC stimulant, GPC Biotech) MX6(apoptosis promotor, MAXIA) CDA-II (apoptosis promotor, Everlife)apomine (apoptosis promotor, ILEX Oncology) SDX-101 (apoptosis promotor,Salmedix) urocidin (apoptosis promotor, Bioniche) rituximab (CD20antibody, Genentech Ro-31-7453 (apoptosis promotor, La Roche) carmustinebrostallicin (apoptosis promotor, Pharmacia) Mitoxantrone β-lapachoneBleomycin gelonin Absinthin cafestol Chrysophanic acid kahweol Cesiumoxides caffeic acid BRAF inhibitors, Tyrphostin AG PD-L1 inhibitors PD-1inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumab sorafenibangiogenesis inhibitors dabrafenib

The following Examples are intended to illustrate the synthesis of arepresentative number of conjugates and the use of these conjugates forthe treatment of cancer. Accordingly, the Examples are intended toillustrate but not to limit the invention. Additional compounds notspecifically exemplified may be synthesized using conventional methodsin combination with the methods described herein.

EXAMPLES Example 1 General Materials and Methods

The antibody used was HuMIgG (Aldrich, 14506). Lutetium-177 was receivedfrom Perkin Elmer as lutetium chloride in a 0.05 N hydrochloric acidsolution.

Analytical HPLC-MS was performed using a Waters Acquity HPLC-MS systemcomprised of a Waters Acquity Binary Solvent Manager, a Waters AcquitySample Manager (samples cooled to 10° C.), a Water Acquity ColumnManager (column temperature 30° C.), a Waters Acquity Photodiode ArrayDetector (monitoring at 254 nm and 214 nm), a Waters Acquity TQD withelectrosparay ionization and a Waters Acquity BEH C18, 2.1×50 (1.7 μm)column. Preparative HPLC was performed using a Waters HPLC systemcomprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/VisibleDetector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prepphenyl or C18 19×100 mm (5 μm) column.

HPLC elution method 1: Waters Acquity BEH C18 2.1 ×50 mm (1.7 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate=0.3 mL/min; initial=90% A, 3-3.5 min=0% A, 4min=90% A, 5 min=90% A.

HPLC elution method 2: Waters XBridge Prep Phenyl 19×100 mm (5 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 13 min=0% A.

HPLC elution method 3: Waters Acquity BEH C18 2.1 ×50 mm (1.7 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate=0.3 mL/min; initial=90% A, 8 min=0% A, 10min=0% A, 11 min=90% A, 12 min=90% A.

HPLC elution method 4: Waters XBridge Prep C18 OBD 19×100 mm (5 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 3 min=80% A, 13min=20% A, 18 min=0% A.

HPLC elution method 5: Waters XBridge Prep C18 OBD 19×100 mm (5 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate: 10 mL/min; initial=90% A, 3 min=90% A, 13min=0% A, 20 min=0% A.

HPLC elution method 6: Waters XBridge Prep C18 OBD 19×100 mm (5 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate: 10 mL/min; initial=75% A, 13 min=0% A, 15min=0% A.

HPLC elution method 7: Waters XBridge Prep C18 OBD 19×100 mm (5 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 12 min=0% A, 15min=0% A.

HPLC elution method 8: Waters XBridge Prep C18 OBD 19×100 mm (5 μm)column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile(0.1% v/v TFA); flow rate: 10 mL/min; initial=90% A, 12 min=0% A, 15min=0% A.

Analytical Size Exclusion Chromatography (SEC) was performed using aWaters system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489UV/Visible Detector (monitoring at 280 nm), a Bioscan Flow Countradiodetector (FC-3300) and TOSOH TSKgel G3000SWxl, 7.8×300 mm column.The isocratic SEC method had a flow rate=1 mL/min, with a mobile phaseof 0.1 M phosphate, 0.6M NaCl, 0.025% sodium azide, pH=7.

MALDI-MS (positive ion) performed using a MALDI Bruker UltraflextremeSpectrometer.

Radio thin-layer chromatography (radioTLC) performed with BioscanAR-2000 Imaging Scanner, carried out on iTLC-SG glass microfiberchromatography paper (Agilent Technologies, SGI0001) plates usingcitrate buffer (0.1 M, pH 5.5).

Example 2 Synthesis of4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoicacid (Compound B)

A bifunctional chelate,4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoicacid (Compound B), was synthesized according to the scheme provided inFIG. 2. To a solution of5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoicacid (DOTA-GA-(tBu)₄, 50 mg, 0.07 mmol) in ACN (2.0 mL) was added DSC(50 mg , 0.21 mmol) followed by pyridine, (0.20 mL, 2.48 mmol). Thereaction was stirred at room temperature for 1 h. To the reactionmixture was added 11-aminoundecanoic acid, (70 mg, 0.36 mmol) followedby PBS solution (1.0 mL) at room temperature. The reaction was stirredfor 72 h at room temperature. The reaction mixture was filtered withsyringe filter and purified directly by Prep-HPLC using method 6 toyield Intermediate 2-A (71 mg, 74.8%).

To a solution of Intermediate 2-A (40 mg , 0.03 mmol), TFP (90 mg , 0.54mmol) and EDC (40 mg, 0.27 mmol) in ACN (1.0 mL) was added pyridine(0.05 mL , 50 mg , 0.62 mmol) at room temperature. The solution wasstirred at room temperature for 24 h. The reaction was purified directlyby Prep-HPLC using method 7 to provide Intermediate 2-B (33 mg, 82.5%)as a wax after concentration using a Biotage V10 Rapid Evaporator.

Intermediate 2-B (33 mg, 0.022 mmol) was dissolved DCM/TFA (1.0 mL/2.0mL) and allowed to stir at room temperature for 24 h. The reaction wasconcentrated by air stream and purified directly by Prep-HPLC usingmethod 8 to yield Compound B (14 mg, 50.0%) as a clear wax afterconcentration. An aliquot was analyzed by HPLC-MS elution method 3;retention time: 4.15 min; MS (positive ESI): found m/z 808.1 [M+H]⁺;C₃₆H₅₄F₄N₅O₁₁ (calc. 808.8).

¹H NMR (600 MHz, DMSO-d₆) δ 7.99-7.88 (m, 1H), 7.82 (t, J=5.5 Hz, 1H),3.78 (broad s, 4H), 3.43 (broad s, 12H), 3.08 (broad s, 4H), 3.00 (m,3H), 2.93 (broad s, 3H), 2.77 (t, J=7.2 Hz, 2H), 2.30 (broad s, 2H),1.88 (broad s, 2H), 1.66 (p, J=7.3 Hz, 2H), 1.36 (m, 4H), 1.32-1.20 (m,9H).

Example 3 Synthesis of4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoicacid (Compound C)

A bifunctional chelate,4-{([2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoicacid (Compound C), was synthesized according to the scheme provided inFIG. 3. To a solution of5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoicacid (DOTA-GA(tBu)₄, 100 mg, 0.143 mmol) in ACN (8.0 mL) was added DSC(73 mg, 0.285 mmol) and pyridine (0.80 mL, 9.89 mmol). The reactionmixture was stirred for 90 min at ambient temperature. This solution wasadded to a semi-solution of amino-PEG3-acid (63 mg, 0.285 mmol in 1.2 mLof DMF) in a 100 mL round bottom flask. After 4 hours at ambienttemperature, the reaction was worked up by concentrating to drynessunder a stream of air. The crude material was purified by HPLC elutionmethod 2 (dissolved the crude in 6 mL of 20% ACN/H₂O). The fractionscontaining product were pooled and concentrated under vacuum and thenco-evaporated with ACN (3×2 mL). Intermediate 1-A was obtained in 82%yield.

To vial containing Intermediate1-A (82 mg, 60 μmol) was added ACN (2mL), NEt₃ (50 μL, 360 μmol, 6 equiv.), HBTU (23 mg, 60 μmol, 1 equiv)and a TFP solution (50 mg, 300 μmol, 5 equiv., dissolved in 250 μL ofACN). The resulting clear solution was stirred at ambient temperaturefor 3 h. The reaction was worked up by concentrating the solution todryness under an air stream and was then diluted with ACN/H₂O (1:1, 3 mLtotal) and purified on preparative HPLC using elution method 4. Thefractions containing product were pooled and concentrated under vacuumand then co-evaporated with ACN (3×2 mL). Intermediate 1-B was obtainedas a clear residue (67 mg, 74% yield).

To a vial containing Intermediate 1-B (67 mg, 64 μmol) was added DCM (2mL) and TFA (2 mL) and the resulting solution was stirred at ambienttemperature for 16 h. Additional TFA (2 mL) was added and the reactionwas stirred at ambient temperature for 6 h. The reaction wasconcentrated to dryness under an air stream with the crude product beingfinally dissolved in ACN/H₂O (1 mL of 10% ACN/H₂O). The crude reactionsolution was then purified by preparative HPLC using elution method 5.The fractions containing product were pooled, frozen and lyophilized.Compound C was obtained as a white solid (36 mg, 63% yield). An aliquotwas analyzed by HPLC-MS elution method 3; retention time: 3.11 min; MS(positive ESI): found m/z 828.4 [M+H]⁺; C₃₄H₅₀F₄N₅O₁₄ (calc. 828.3).

¹H NMR (DMSO-d₆, 600 MHz) δ 7.97-7.91 (m, 2H), 3.77 (t, 2H, J=6.0 Hz),3.58-3.55 (m, 2H), 3.53-3.48 (m, 8H), 3.44-3.38 (m, 10H), 3.23-3.08 (m,11H), 3.02 (t, 2H, J=6.0 Hz), 2.93 (broad s, 4H), 2.30 (broad s, 2H),1.87 (broad s, 2H).

Example 4 Synthesis of [¹⁷⁷Lu]-Compound A-Human-IgG

The compound Compound A (1.34 μmoles) was dissolved in sodium acetatebuffer (20 μL, pH 6.5) and added to a solution containing the antibodyHuman-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After45 minutes at ambient temperature the antibody conjugate product waspurified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH6.5, 1 mM ascorbic acid). MALDI-TOF-MS (positive ion): CompoundA-Human-IgG: found m/z 150360 [M+H]⁺; Human-IgG: found m/z 148339[M+H]⁺.

As a typical reaction, Lu-177 (1.1 mCi, 5 μL) iswas added to a solutionof Compound A-Human-IgG (90 μg in acetate buffer (pH 6.5) and ascorbicacid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reactionwas incubated at 37° C. for 90 minutes. The crude product,[¹⁷⁷Lu]-Compound A-Human-IgG, was purified via a Sephadex G-50 resinpacked column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid.RadioTLC radiochemical purity: 98%; radiochemical yield: 45%; specificactivity: 15.1 mCi/mg.

Example 5 Synthesis of [¹⁷⁷Lu]-Compound B-Human-IgG

The compound Compound B (1.17 μmoles) was dissolved in sodium acetatebuffer (0.117 mL, pH 6.5). An aliquot of the Compound B solution (2 μL,10 nmoles) was added to a solution containing the antibody Human-IgG(6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambienttemperature the antibody conjugate product was purified via a SephadexG-50 resin packed column. The antibody conjugate Compound A-Human-IgGwas eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS(positive ion): Compound B-Human-IgG found m/z 149949 [M+H]⁺; Human-IgGfound m/z 148540 [M+H]⁺.

As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to asolution of Compound B-Human-IgG (100 μg in acetate buffer (pH 6.5) andascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabelingreaction was incubated at 37° C. for 30 minutes. The crude product,¹⁷⁷Lu-Compound B-Human-IgG, was purified via a HPLC SEC column (1mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) andconcentrated by ultrafiltration (Vivaspin, 10 kDa,). RadioTLCradiochemical purity: 98%; radiochemical yield: 51%; specific activity:9.68 mCi/mg.

Example 6 Synthesis of [¹⁷⁷Lu]-Compound C-Human-IgG

The compound Compound C (0.96 μmoles) was dissolved in sodium acetatebuffer (95 μL, pH 6.5). An aliquot of the Compound C solution (2 μL, 20nmoles) was added to a solution containing the antibody Human-IgGantibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour atambient temperature the antibody conjugate product was purified via aSephadex G-50 resin packed column. The antibody conjugate CompoundC-Human-IgG was eluted from the column with acetate buffer (pH 6.5).MALDI-TOF-MS (positive ion): Compound C-Human-IgG: found m/z 150095[M+H]⁺; Human-IgG: found m/z 148540 [M+H]⁺.

As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to asolution of Compound C-Human-IgG (100 μg in acetate buffer (pH 6.5) andascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). Theradiolabeling reaction was incubated at 37° C. for 30 minutes. The crudeproduct, [¹⁷⁷Lu]-Compound C-Human-IgG, was purified via a HPLC SECcolumn (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbicacid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,). RadioTLCradiochemical purity: 98%; radiochemical yield: 37%; specific activity:9.99 mCi/mg.

Example 7 Pharmacokinetic and Metabolism Studies of Human-IgG-BasedCompounds

Groups of 4 or 5 mice (normal CD-1 or athymic CD-1 nude) were injectedintravenously with approximately 15 microcuries of radiolabelled testcompound. Test compounds with various linkers were synthesized andradiolabelled with lutetium-177. For pharmacokinetic studies, animalswere sacrificed at specific time points, and blood and tumor (whenapplicable) were analyzed for total radioactivity. For metabolismstudies, animals were placed in metabolic cages (4-5 per cage) for urineand feces collection every 24 hours for up to 7 days. The radioactivecontent of urine and feces samples was quantified and converted to totalurine or feces output based on weight. Excretion profiles for urine,feces, or total excretion (urine+feces) were generated by plottingcumulative % injected dose (%ID) over time.

Non-targeted human IgG antibodies were used for metabolic excretionstudies in order to demonstrate that the alterations in radioactivityexcretion profiles directed by conjugation with linker Compound B andCompound C is a general process. The human IgG preparation usedconsisted of a purified mixture of all IgG isotypes (IgG1-4).

The metabolic excretion profile of a non-targeted human IgG antibodyconjugates [¹⁷⁷Lu]-Compound B-HuMIgG, and [¹⁷⁷Lu]-Compound C-HuMIgG werecompared to [¹⁷⁷Lu]-Compound A-HuMIgG. The [¹⁷⁷Lu]-Compound A-HuMIgG wasexcreted slowly with just 13% of the injected dose (ID) eliminated over7 days by low level urinary excretion. Compounds B and C directeddistinctly different excretion routes and increased the amount totalexcretion of radioactivity over a 7 day period when compared to CompoundA-HuMIgG. [¹⁷⁷Lu]-Compound B-HuMIgG was eliminated through the feces and[¹⁷⁷Lu]-Compound C-HuMIgG elimination was roughly equally dividedbetween the urine and feces. Therefore, it was found that while thelinker type impacted the route, rate, and extent of compound excretion(FIG. 4), it did not alter the overall blood pharmacokinetics of thetotal radioactivity associated with the radioimmunoconjugate. It wasalso observed that the improved excretion profile of the Compound B orCompound C when conjugated to antibodies is a general and reproducibleeffect.

Example 8 Synthesis of [²²⁵Ac]-Compound A-Human-IgG

The compound Compound A (1.34 μmoles) was dissolved in sodium acetatebuffer (20 μL, pH 6.5) and added to a solution containing the antibodyHuman-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After45 minutes at ambient temperature the antibody conjugate product waspurified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH6.5, 1 mM ascorbic acid). MALDI-TOF-MS (positive ion): CompoundA-Human-IgG: found m/z 150360 [M+H]⁺; Human-IgG: found m/z 148339[M+H]⁺.

As a typical reaction, Ac-225 (1.1 mCi, 5 μL) is added to a solution ofCompound A-Human-IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid(1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction isincubated at ambient temperature (e.g., 20-25° C.) for 90 minutes. Thecrude product, [²²⁵Ac]-Compound A-Human-IgG, is purified via a SephadexG-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mMascorbic acid).

Example 9 Synthesis of [²²⁵Ac]-Compound B-Human-IgG

The compound Compound B (1.17 μmoles) was dissolved in sodium acetatebuffer (0.117 mL, pH 6.5). An aliquot of the Compound B solution (2 μL,10 nmoles) was added to a solution containing the antibody Human-IgG(6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambienttemperature the antibody conjugate product was purified via a SephadexG-50 resin packed column. The antibody conjugate Compound A-Human-IgGwas eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS(positive ion): Compound B-Human-IgG found m/z 149949 [M+H]⁺; Human-IgGfound m/z 148540 [M+H]⁺.

As a typical reaction, the Ac-225 (1.1 mCi, 5 μL) is added to a solutionof Compound B-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbicacid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabelingreaction is incubated at ambient temperature (e.g., 20-25° C.) for 30minutes. The crude product, [²²⁵Ac]-Compound B-Human-IgG, is purifiedvia a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10kDa).

Example 10 Synthesis of [²²⁵Ac]-Compound C-Human-IgG

The compound Compound C (0.96 μmoles) was dissolved in sodium acetatebuffer (95 μL, pH 6.5). An aliquot of the Compound C solution (2 μL, 20nmoles) was added to a solution containing the antibody Human-IgGantibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour atambient temperature the antibody conjugate product was purified via aSephadex G-50 resin packed column. The antibody conjugate CompoundC-Human-IgG was eluted from the column with acetate buffer (pH 6.5).MALDI-TOF-MS (positive ion): Compound C-Human-IgG: found m/z 150095[M+H]⁺; Human-IgG: found m/z 148540 [M+H]⁺.

As a typical reaction, the Ac-225 (1.1 mCi, 5 μL) is added to a solutionof Compound C-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbicacid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabelingreaction is incubated at ambient temperature (e.g., 20-25° C.) for 30minutes. The crude product, [²²⁵Ac]-Compound C-Human-IgG, is purifiedvia a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10kDa,).

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features herein before set forth.

What is claimed is:
 1. A compound having the structure:A-L¹-(L²)_(n)-B   Formula I wherein A is chelating moiety or a metalcomplex thereof; L¹ is optionally substituted C₁-C₆ alkyl, substitutedC₁-C₆ heteroalkyl, substituted aryl or heteroaryl; B is a is atherapeutic moiety, a targeting moiety, or cross-linking group, or apharmaceutically acceptable salt thereof; n is 1-5; each L²,independently, has the structure:(—X¹-L³-Z¹—)   Formula II wherein is X¹ is C═O(NR¹), C═S(NR¹),OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹), —CH2PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹) ,O, NR¹ and R¹ is H or optionally substituted C₁-C₆ alkyl or optionallysubstituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl; L³ isoptionally substituted C₁-C₅₀ alkyl or optionally substituted C₁-C₅₀heteroalkyl or C₅-C₂₀ polyethylene glycol; Z¹ is CH₂, C═O, C═S, OC═O,NR¹C═O, NR¹; and R¹ is a hydrogen or optionally substituted C₁-C₆ alkyl,pyrrolidine-2,5-dione.
 2. The compound in claim 1, wherein said thechelating moiety is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α, α′, α″,α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane),DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid),DO3AM-acetic acid(2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)aceticacid), DOTA-GA anhydride(2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP(1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid,DOTA-4AMP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonicacid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP(1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA(1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid),PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaaceticacid), H₄octapa(N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diaceticacid), H₂dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane),H6phospa(N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane),TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P(tetraazacyclododecane dimethanephosphonic acid), HP-DO3A(hydroxypropyltetraazacyclododecanetriacetic acid), EDTA(ethylenediaminetetraacetic acid), Deferoxamine, DTPA(diethylenetriaminepentaacetic acid), DTPA-BMA(diethylenetriaminepentaacetic acid-bismethylamide), or porphyrin. 3.The compound of claim 2, wherein the chelating moiety has the structure:

Wherein Y¹ is —CH₂OCH₂(L²)_(n)—B, C═O(L²)_(n)-B, or C═S(L²)_(n)-B and Y²is —CH₂CO₂H; Wherein Y¹ is H, Y² is L¹-(L²)_(n)-B.
 4. The compound ofclaim 1, wherein L¹ has the structure:

wherein R² is optionally substituted hydrogen or —CO₂H
 5. The compoundof claim 4, wherein said metal complex can be have a metal selected fromBi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, Sm, a lanthanide,or an actinide, for use as imaging or therapeutic agents and may beselected from radionuclides suitable for complexing to a compound offormula (I) include ₄₇Sc, ⁵⁵Co, ₆₀Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ₆₇Cu, ⁶⁶Ga,⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁹⁷Ru, ^(99m)Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In,^(117m)Sn, ¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²⁰¹Tl,²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th.
 6. The compound of claim1, wherein B is a therapeutic moiety or targeting moiety.
 7. Thecompound of claim 6, wherein the therapeutic moiety or targeting moietyis an antibody, an antigen-binding fragment thereof or other targetingprotein such as nanobodies, affibodies, and consensus sequences fromFibronectin type III domains.
 8. The compound of claim 7, wherein theantibody, or an antigen-binding fragment thereof specifically bindsinsulin-like growth factor-1 receptor (IGF-1R).
 9. The compound of claim1, wherein the cross-linking group is an amino-reactive, amethionine-reactive, or a thiol-reactive cross-linking group.
 10. Thecompound of claim 9, wherein the amino-reactive, methionine-reactive, orthiol-reactive cross-linking group comprises an activated ester such asa hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester,4-nitrophenol ester or an imidate, anhydride, thiol, disulfide,maleimide, azide, alkyne, strained alkyne, strained alkene, halogen,sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine,tetrazine, isothiocyanate, or oxaziridine.
 11. The compound of claim 9or 10, wherein the cross-linking group is selected from the groupconsisting of:


12. The compound of claim 3, wherein Y¹ is H.
 13. The compound of claim1, wherein X¹ is C═O(NR¹) and R¹ is H.
 14. The compound of claim 1,wherein Z¹ is —CH₂.
 15. The compound of claim 1, wherein L² has n valueof
 1. 16. The compound of claim 1, wherein the compound is selected fromthe group consisting of:


17. The compound of claim 5, wherein the metal is a radionuclide. 18.The compound of claim 17, wherein the radionuclide is In¹¹¹.
 19. Thecompound of claim 17, wherein the radionuclide is Ga⁶⁸.
 20. The compoundof claim 1, wherein the metal is an alpha-emitting radionuclide.
 21. Thecompound of claim 20, wherein the radionuclide is Ac²²⁵ or the progeny(daughter isotopes) thereof.
 22. A pharmaceutical composition comprisinga compound of claim 1 and a pharmaceutically acceptable excipient.
 23. Amethod of radiation treatment planning and/or radiation treatment andthe method comprising administering to a subject in need thereof acompound of claim
 1. 24. The method of claim 23, the method comprisingadministering to a subject in need thereof a first dose of a compound ofclaim 1 in an amount effective for radiation treatment planning,followed by administering subsequent doses of a compound of claim 1 in atherapeutically effective amount.
 25. The method of claim 24, whereinthe compound administered in the first dose and the compoundadministered in the second dose are the same.
 26. The method of claim24, wherein the compound administered in the first dose and the compoundadministered in the second dose are different.
 27. The method of claim24, wherein the cancer is a solid tumor or hematologic (liquid) cancer.28. The method claim 27, wherein the solid tumor cancer is breastcancer, non-small cell lung cancer, small cell lung cancer, pancreaticcancer, head and neck cancer, prostate cancer, colorectal cancer,sarcoma, adrenocortical carcinoma, Ewing's Sarcoma, multiple myeloma, oracute myeloid leukemia.
 29. The method of claim 28, further comprisingadministering an antiproliferative agent, radiation sensitizer, or animmunoregulatory or immunomodulatory agent.
 30. The method of claim 29,wherein the compound of claim 1 and the antiproliferative agent orradiation sensitizer are administered within 28 days of each other. 31.The method of claim 29, wherein the compound of claim 1 and animmunoregulatory or immunomodulatory agent are administered within 90days of each other.